This work is directed toward the identification, purification and characterization of enkephalin degrading enzymes, enkephalinases, in mammalian brain. Enkephalinases A1, A2 and B will be solubilized from rat brain membranes and purified by a variety of chromatographic techniques. Pure enzyme preparations will be characterized with respect to their activity toward enkephalin analogs and other neuropetide transmitters. Antibodies to these enzymes will be used as specific probes for enzyme identification, distribution and turnover. Fluorgenic histochemical substrates will be developed as a means to specifically measure and localize enkephalinases. We will conduct studies on the relationship between enkephalin degradation and opiate receptor occupancy. By means of cross-linking studies, we will determine the architectural relationship of enkephalinases and opiate receptor. The role of enkephalinases in the morphine addiction, tolerance and withdrawal syndrome will be studied. The scientific disciplines involved in these studies are Biochemistry, neurobiology, neurochemistry, neuroimmunology neuropharmacology, neurohistochemistry and cell biology.